Date of Award

Summer 8-15-2015

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The Gram-negative bacterium uropathogenic Escherichia coli (UPEC) is the causative agent of up to 85% of community-acquired urinary tract infections (UTIs). This thesis work aimed to better define interactions between UPEC and host antimicrobial peptides (AMPs). The bacterial outer membrane is constantly interacting with the external environment, and contains many integral proteins with defined roles in pathogenesis. The E. coli outer membrane protein OmpA is a highly conserved, integral beta-barrel membrane protein with four extracellular loops. We found that the loops of OmpA individually influence binding and invasion of UPEC during infection. Loop 2 appears to be essential for binding to the host epithelium. These effects are seen in vitro as well as in a murine model of UTI, where the pathogenic cascade is severely interrupted when loop 2 is absent. The loop 2 mutant forms very few intracellular bacterial communities (IBCs, a hallmark of UPEC UTI) and fails to cause robust acute infection. These phenotypes are not due to alterations in membrane permeability, growth, or the indispensable adhesive virulence organelle termed type 1 pili.

AMPs are small proteins expressed by innate immune cells and epithelial cells, including those of the urinary tract. Although we predicted that the murine cathelicidin, termed CRAMP, would play an important role in urinary tract defense against UPEC, we instead found that CRAMP-deficient mice exhibit attenuated UTIs. These mice have lower bladder bacterial loads at all examined time points, fewer IBCs, less inflammation and inflammation-related bladder damage, and resolve infection faster. Early differences in bacterial binding to wild-type and mutant bladders are not attributable to modifications in bacterial adhesive factors, instead pointing to altered host uroepithelium in CRAMP-deficient mice. Furthermore, cathelicidin does not appear to be expressed within the urinary tract at concentrations necessary to cause bacterial cell death in vitro. We conclude that cathelicidin is essential for normal maintenance of the uroepithelium, and for the promotion of innate immune responses to UPEC. We will continue to examine this potential role for CRAMP as a driver of the immune response during UTI.

Finally, we were surprised to find that UPEC growth is augmented after exposure to sub-lethal concentrations of CRAMP, and UPEC specifically upregulate a defined set of genes in this response. While these genes are also encoded by non-pathogenic E. coli, the same transcriptional response is not observed. Most interestingly, the majority of these genes are involved in small molecule transport or metabolism, implying that UPEC are actively sensing the peptide, upregulating pathways that may allow for uptake of the AMP, and perhaps converting acquired AMP into useful product. This model is consistent with the increased UPEC growth seen after exposure to CRAMP, and suggests that UPEC are better adapted to strategically utilize molecules in order to thrive in a limiting environment. Future experiments will characterize the roles of these regulated genes in vitro after exposure of UPEC to other AMPs, and during infection.

Language

English (en)

Chair and Committee

David A Hunstad

Committee Members

Michael Caparon, James Fleckenstein, Jeffrey Henderson, Katherine Henzler-Wildman, Scott Hultgren

Comments

Permanent URL: https://doi.org/10.7936/K7V12327

Download

Share

COinS